Difference: ProtocolsAntibioticStockSolutions (47 vs. 48)

Revision 482018-10-25 - GabrielSuarez

META TOPICPARENT	name="ProtocolsGeneGorgingMarker"

Media Amendment Stock Solutions

Reagent	Abb	Stock Conc.	Working Conc.	Dilution	Solvent	MSDS	Notes
Ampicillin	Amp	100 mg/mL	100 µg/mL	1,000×	ddH₂O	link
Anhydrotetracycline	ATC	0.1 mg/mL	100 ng/mL	1,000×	DMSO	link	Can also make a 1mg/mL stock and dilute 1:10 for a final concentration of 0.1mg/mL
3′-Azido-2′,3′-dideoxythymidine	AZT	10 mg/mL	200 µg/mL	50×	ddH₂O	link	Can substitute pharma grade Zidovudine. Soluble up to 20 mg/mL in ddH₂O
Bromo-chloro-indolyl-galactopyranoside	X-gal	40 mg/mL	80 µg/mL	500×	Dimethylformamide (DMF)	link	X-gal is a histochemical substrate for β-galactosidase, which cleaves X-Gal to yield an insoluble blue precipitate.
Carbenicillin	Crb	100 mg/mL	100 µg/mL	1,000×	50% EtOH	link
Chloramphenicol	Cam	20 mg/mL	20 µg/mL	1,000×	EtOH	link
Cycloheximide	Chx	100 mg/mL	100 µg/mL	1,000×	100% DMSO	link	Anti-fungal, toxic to humans, use PPE!
D-Cycloserine	Dcs	4 mg/mL	4 µg/mL	1,000×	ddH₂O	link	Degrades noticeably over the course of 1 week at 4°C
Diaminopimelic acid	DAP	100 mM	0.3 mM	333×	ddH₂O	link	Use 3 µl stock per 1 ml of media. Dissolve with overnight shaking.
Erythromycin	Ery	250 mg/mL	250 µg/mL E. coli 10 µg/mL Lactobaccillus sp.	1,000x	EtOH	link	May crystallize when added to liquid culture. Still works. Selection strength may vary in non-model bacteria
Gentamicin	Gen	5 mg/mL	5 µg/mL	1,000×	ddH₂O	link	For non-canonical experiments: Use 20 µg/mL in culture from 20mg/mL 1000× stock
Isopropyl β-D-1-thiogalactopyranoside	IPTG	0.1 M	0.1 mM	1,000×	ddH₂O	link	Light sensitive, MW: 238.31 g/mol

Changed:

<
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Kanamycin

Kan

50 mg/mL

50 µg/mL

1,000×

ddH₂O

link

Plates lose sensitivity after 2 weeks

>
>

Kanamycin

Kan

50 mg/mL

50 µg/mL

1,000×

ddH₂O

link

Plates start to lose sensitivity after 2 weeks; recommended 4C storage time is ~1 month

Nalidixic Acid	Nal	30 mg/mL	30 µg/mL	1,000×	1M NaOH	link
Novobiocin	Nov	200 mg/mL	200 µg/mL	1,000×	ddH₂O	link
Rifampicin	Rif	50 mg/mL	100 µg/mL	500×	MeOH + drops of 10M NaOH	link	Toxic to humans, use PPE!
Spectinomycin	Spec	60 mg/mL	60 µg/mL	1,000×	ddH₂O	link	Common to see background growth of cells on selective plates
Streptomycin	Str	100 mg/mL	100 µg/mL	1,000×	ddH₂O	link
Tetracycline	Tet	10 mg/mL	10 µg/mL	1,000×	70% EtOH	link	Light sensitive, stability < 2 weeks
Zeocin	Zeo	10 mg/mL	10 µg/mL	1,000×	ddH₂O	link	Some labs report using a higher concentration for selection. 10 µg/mL is specifically used for growth of amberless strain A37

Working concentrations are for liquid or solid media.

Preparation

Generally, prepare 30-50 mL of solution in a 50 mL conical tube. Then, filter sterilize solutions by pushing them through a 50 mL syringe fitted with a 0.22 µm filter made of a material appropriate for the solvent. Aliquot into 1.5 mL portions in 1.7 mL tubes using the repeat pipettor. Label with a permanent marker (one that won't wash off with ethanol). Store at -20°C in the dark.

Other considerations

Rifampicin is TOXIC to humans, light-sensitive, and degrades in solution rather quickly. It needs to be stored wrapped in foil and replaced every 2 months.
Ampicillin degrades quickly in both plates and stock solutions. Culture plates with Amp can be stored at 4°C for about 2 weeks. Stock solutions can be stored at 4°C for 2 weeks but can last as long as 4-6 months when stored at -20°C. In general, use carbenicillin for ampicillin selection, because it is more chemically stable.
D-cycloserine is best used within two days of pouring. A single small-scale experiment found 6-10x as many E. coli colonies on 8-day old MG-Dcs plates that had spent 3 of those days at -20°C and 5 at 4°C, compared to what was found on 1 day old MG-Dcs.
Kanamycin plates only last a few weeks at 4°C; best to make fresh Kan plates every 2 weeks.

Non-canonical Amino Acid Stock Solutions

Amino Acid (Abbreviation)	CAS Number	Solvent	MW (g/mol)	[Stock]	[Stock] (mg/mL)	[Culture]	Notes
Y	60-18-4	Water	181.19	10mM	1.812	250µM	Requires heating to 70ºC to dissolve Stock precipitates out of solution at or below room temperature
IodoY	70-78-0	Water	307.09	10mM	3.071	250µM	Dissolves at room temperature with ~1 hour of shaking
AminoY (FAA)	300-34-5		196.203
AminoY (HS)	23279-22-3	Water	287.14	10mM		250µM	Dissolves readily without shaking or heat
BromoY	38739-13-8	Water	260.0846	10mM		250µM
ChloroY	7423-93-0	Water	215.63	10mM		250µM
NitroY	621-44-3	Water	226.2	10mM	2.262	250µM	Will dissolve with heating to 50ºC, no shaking
ONBY (FAA)	184591-51-3		316.31				Light sensitive
ONBY (S)	207727-86-4	50% DMSO 50% Water (exothermic)	352.77	50 mM		250µM	Light sensitive Requires adding OH^- to dissolve
AzF	33173-53-4	10% DMSO 90% Water	206.204	10mM	2.062	250µM	Light sensitive Requires heating and shaking to dissolve
CNF	167479-78-9	Water	190.2	10mM	1.902	250µM	Dissolves at room temperature with ~15 minutes of shaking
L-DOPA	59-92-7	Water	197.1879	10mM	1.972	250µM	Oxidizes quickly and turns black in solution
5HTP	4350-09-8	Water	220.2246	10mM	2.202	250µM

FAA: free amino acid
S: salt
HS: hydrated salt

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